Sortase A Inhibitors: Recent Advances and Future Perspectives

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Sortase A Inhibitors: Recent Advances and Future Perspectives Stella Cascioferro,*,†,‡ Demetrio Raffa,† Benedetta Maggio,*,† Maria Valeria Raimondi,† Domenico Schillaci,† and Giuseppe Daidone† †

Downloaded by UNIV OF SOUTH DAKOTA on September 15, 2015 | http://pubs.acs.org Publication Date (Web): August 17, 2015 | doi: 10.1021/acs.jmedchem.5b00779

Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Sezione di Chimica e Tecnologie Farmaceutiche, Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy ‡ IEMEST, Istituto Euromediterraneo di Scienza e Tecnologia, Via Emerico Amari, 123, 90139 Palermo, Italy ABSTRACT: Here, we describe the most promising small synthetic organic compounds that act as potent Sortase A inhibitors and cater the potential to be developed as antivirulence drugs. Sortase A is a polypeptide of 206 amino acids, which catalyzes two sequential reactions: (i) thioesterification and (ii) transpeptidation. Sortase A is involved in the process of bacterial adhesion by anchoring LPXTG-containing proteins to lipid II. Sortase A inhibitors do not affect bacterial growth, but they restrain the virulence of pathogenic bacterial strains, thereby preventing infections caused by Staphylococcus aureus or other Gram-positive bacteria. The efficacy of the most promising inhibitors needs to be comprehensively evaluated in in vivo models of infection, in order to select compounds eligible for the treatment of bacterial infections in humans.

1. INTRODUCTION The development of antibiotics that specifically target bacteria cells and are therefore endowed with a good profile of safety and tolerability has highly contributed to demographic growth and the improvement of the quality of life in the last 75 years. However, the emergence of multidrug-resistant Grampositive and Gram-negative bacterial strains has limited and continues to limit the clinical efficacy of most of the currently marketed antibiotics. Drug-resistant bacteria are responsible for 5,000 deaths per year in the UK, and 25,000 deaths per year in Europe. It is estimated that at least two million people in the United States are infected each year, and 23,000 of them die because the underlying bacterial strains are not susceptible to treatment by any of the current antibiotics.1,2 The World Health Organization, in a recent report on antimicrobial resistance in common bacterial pathogens, highlights that a postantibiotic era may start the 21st century.3 Unfortunately, the current investments of drug companies in the development of new antibiotics are largely reduced in spite of the high medical need. In addition to a high selectivity index (i.e., the ratio between toxic and effective doses), a new ideal antimicrobial agent should harbor three main features: (i) a low selectivity pressure to avoid the rise in antibiotic-resistance strains; (ii) the ability to kill pathogens without harming the microbiota; and (iii) the ability to counteract natural forms of resistance exhibited by multistratified microbial populations growing on surfaces, the so-called biofilms. Antivirulence agents meet these requirements. Over the past decade, many studies have focused on agents that specifically target the molecular determinants of virulence with no bactericidal effect and no effect on bacterial growth. In contrast, conventional antibiotics © XXXX American Chemical Society

hit targets associated with cell death and the development of drug resistance is always advantageous to the microorganism.4 Antivirulence drugs disarm pathogenic bacteria rather than kill them, and therefore, they exert low selective pressure to promote the development of antibiotic resistance.5 The increased knowledge of mechanisms and processes associated with virulence in several pathogenic strains may lay the groundwork for the study and the development of an antivirulence approach to fight the pathogens.6−8 A fundamental and common step in the pathogenic action of Gram-positive and Gram-negative bacteria is the adhesion to the host tissue mediated by a direct and specific interaction between bacterial surface molecules and host ligands. As a consequence of this interaction, bacteria are not mechanically removed from the host.9 Interference with adhesion may provide an efficient strategy for the prevention or treatment of bacterial infections. In this field, important advancements have been made in serious Gram-negative pathogens including uropathogenic Escherichia coli and Pseudomonas aeruginosa.10,11 Here, we focus on Staphylococcus aureus, a Gram-positive bacterium that may cause nosocomial infections such as bacteremia, endocarditis, osteomyelitis, pneumonia, and infections of the skin and soft tissues.12,13 Methicillin-resistant Staphylococcus aureus has been considered by the Antimicrobial Availability Task Force of the Infectious Diseases Society of America, as one of six high-priority dangerous ESKAPE Received: May 22, 2015

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DOI: 10.1021/acs.jmedchem.5b00779 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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pathogens (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter).14−16

the protein and are essential for the anchoring of MSCRAMMs to the cell wall. The first step in the adhesion process is the “recognition reaction” between Srt A and the MSCRAMM by the LPXTG motif (Figure 2a). After recognition, SrtA catalyzes two sequential reactions: (i) thioesterification and (ii) transpeptidation. The enzyme first cleaves the LPXTG pentapeptide sorting motif between Thr and Gly residues, thereby causing the formation of a thioester acyl-enzyme intermediate (Figure 2b). The latter is then resolved through a SrtA-mediated transpeptidation, with ensuing formation of an amide linkage of the C-terminal Thr of the protein to pentaglycine cross-bridges in S. aureus (Figure 2c). It has been suggested that SrtA uses the cell wall precursor lipid II and that the proteins linked to lipid II are subsequently incorporated into the peptidoglycan.20 There are about 20 staphylococcal proteins characterized by a C-terminal LPXTG motif, including protein A (Spa), two fibronectin binding proteins (FnbpA and FnbpB), two clumping factors (ClfA and ClfB), a collagen-binding protein (Cna), and three serineaspartate repeat proteins (SdrC, SdrD, and SdrE).21 SrtA is considered as a good target for new antivirulence drugs that interfere with bacterial adhesion,20 and therefore, its relationship to bacterial pathogenesis has been extensively investigated. Mazmanian et al. in 2000 studied srtA knockout mutants of S. aureus that are defective in surface expression of different LPXTG motif proteins. Remarkably, srtA mutants did not cause renal abscesses and acute infection in mice.22 These data have been confirmed by studies carried out in several animal models of infection.23 Inhibition of SrtA also causes loss of binding activity to IgG, fibronectin, and fibrinogen, with consequent attenuation of bacterial virulence24 and reduction of biofilm formation in some staphylococcal strains.25 Similarly to what was described for S. aureus, the loss of transpeptidase activity of sortase in other Gram-positive pathogens, including L. monocytogenes,26 S. pneumoniae,27 S. mutans,28 and Streptococcus suis29 results in virulence attenuation in animal infection models because of a failure to express LPXTG surface proteins.


2. SORTASE A AND BACTERIAL VIRULENCE Adhesion to host tissue is a critical step for bacterial virulence and involves a vast array of cell wall proteins and protein complexes in both Gram-positive and Gram-negative bacteria.9,17 In Gram-negative bacteria, pili play a major role in adhesion. Pili are structures composed of several hundreds of small subunits of 15−25 kDa, called pilins. Pilins are α,β proteins characterized by very long N-terminal α-helix domains that self-assemble in a head-to-tail manner to form a complete pilus. In contrast, adhesion of Gram-positive bacteria involves proteins with structural motifs similar to those of pilin components (Figure 1), denominated “microbial surface

Figure 1. Common structural motifs (S, W, M, and C) present in the MSCRAMMs and pilin components.19

components recognizing adhesive matrix molecules” (MSCRAMMs).18,19 These surface proteins are covalently linked to the peptidoglycan by transpeptidase sortase A (SrtA), and they recognize important host extracellular proteins such as fibrinogen, fibronectin, and collagen. In particular, as shown in Figure 1, the cell wall sorting motifs W, which include a LPXTG (leucine, proline, any amino acid, threonine and glycine) sequence at the carboxyl terminal region, the hydrophobic region M, and the ending C with a tail of positively charged amino acids, operate as a sorting signal of

Figure 2. Mechanism of action of SrtA: (a) recognition process, (b) thioesterification reaction, and (c) transpeptidation reaction. B



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In particular, the β6/β7 loop is essential for the catalytic activity of the enzyme, and it undergoes a conformational change once SrtA binds with the sorting signal passing from a disordered and open conformation to an ordered and shortened conformation of a 310 helix free of residues Thr156 and Val161, which are incorporated into strand β6 (Figure 5). Enzymes belonging to the family of sortase possess three conserved residues in their active sites, His120, Cys184, and Arg197, the importance of which in catalysis has been widely demonstrated (Figure 6).33,35−37 Accordingly, the substitution of these amino acids with alanine abolishes sortase activity in vivo and in vitro.38 These three amino acid residues represent a catalytic triad similar to that exhibited by other enzymes such as proteases, amidases, esterases, acylases, lipases, and β-lactamases. In particular, the Cys-His-Asp triad is used by several classes of cysteine proteases, amidases, and papain; Srt A uses a similar triad in which the residue asparagine is substituted by an arginine. Regardless of its structure, the triad is formed by three common elements: an acid, a base, and a nucleophile residue. The acid residue (commonly glutamate or aspartate) aligns and polarizes the base (usually histidine) that activates the nucleophile (often serine or cysteine, occasionally threonine) as shown in Figure 7. The function of the triad is therefore to increase the reactivity of the nucleophile residue for efficient catalysis by reducing its pKa.39 A comparison between the catalytic triad of SrtA and papain has been published.31,33 In papain, a thiolate-imidazolium ion pair between the side chains of His159 and Cys25 (papain numbering, PDB: 1PPN) holds the cysteine side chain in an active configuration.40 In the structure of SrtA without substrate, the side chains of His120 and Cys25 (SrtA numbering, PDB: 2KID30) project far from each other without interacting.31 To enable sulfhydryl proton extraction and nucleophile attack, a conformational rearrangement, favored by a substrate-induced activation, must occur in these side chains. In SrtA, the active site residue Cys184 is essential for the catalytic activity of the enzyme because it is responsible for a nucleophilic attack of the thiol group on the carbonyl group of the Thr, causing the formation of a thioester acyl enzyme intermediate, which is then captured by the peptidoglycan through an amide linkage of the C-terminal Thr of the protein to the amino group of the pentaglycine cross-bridge.31 Arg197 seems to deprotonate Cys184 and the Gly5 chain of lipid II41,34 and to stabilize the tetrahedral oxyanion transition state prior to the formation of the acyl-enzyme intermediate.42 His120 has been initially suggested to have a specific role in the activation of Cys184 via the formation of an imidazoliumthiolate ion pair.35 However, further studies have demonstrated that His120 functions as a general base during the catalysis.39 The two residues Cys184 and His120 found in the active site of SrtA in S. aureus are highly conserved in other Gram-positive bacteria, including Streptococcus pyogenes,43 Streptococcus pneumonia,44 and Lysteria monocytogenes.45 The importance of several amino acids residues of the β6/β7 loop, such as Val168, Leu169, Ala104, Ala118, Leu97, for the enzymatic activity has been highlighted by Suree et al.30 who performed in situ mutagenesis studies. The V168A, L169A, A104G, A118G, and L97V mutants were several times less active than the wild-type protein.


3. SORTASE A STRUCTURE The structure of the S. aureus SrtA, the prototypical member of the sortase enzyme family, is well established (Figure 3).30−33

Figure 3. Ribbon drawing of the structure of the Staphylococcus aureus sortase A-complex (PDB: 2KID).30 The coils are in green, the helices in blue, the strands in red, the ligand in yellow, and the calcium ion in black.

SrtA is a polypeptide of 206 amino acids, which consists of two regions: (i) an unstructured amino-terminal tail containing a stretch of nonpolar residues that embed the protein in the membrane and (ii) an autonomously folded catalytic domain that performs the transpeptidation reaction. This reaction involves the cleavage of the C-terminal sorting signal LPXTG motif of surface protein precursors between threonine T and glycine G residues (Figure 4, step 1). A covalent thioester bond between the thiol group Cys184 of the enzyme and the carboxyl group of the Thr residue of the substrate is thus formed. This transient thioester acyl-enzyme intermediate I (Figure 4, step 2) undergoes a nucleophilic attack from the amino group of the Gly, which forms an amide bond between the carboxylic group of Thr and the amino group of the cell wall cross-bridge; through this mechanism, the substrate protein covalently links to the peptidoglycan (Figure 4, step 2). NMR and X-ray studies of a recombinant SrtAΔN59 have revealed that it assumes an eight-stranded β-barrel fold34 and that the enzyme recognizes the LPXTG sorting signal through a large groove that leads into the active site. Strands β4 and β7 are in the floor of the groove, while the loops β6/β7, β7/β8, β3/β4, and β2/H1 form the walls.30 C




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Figure 4. Possible mechanism of transpeptidation.

Figure 6. Representation of the most important amino acids in the active site of SrtA (PDB: 1KID).30 Figure 5. Superimposition of the NMR structure of the apo-SrtA (2KID,30 hot pink) and StrA-LPAT complexes (1IJA,31 cyan): (a) whole structures and (b) details of the structures in which the conformational change in loops β6/β7 and β7/β8 induced by the substrate are highlighted.

4. SORTASE A INHIBITORS Several strategies have been developed for the identification and characterization of new sortase A inhibitors. These include screening of natural product or small compound libraries, which led to the discovery of the natural products β-sitosterol3-O-glucopyranoside 1,46 berberine chloride 2,47 bis(indole)alkaloid 3,48 isoaaptamine 4,49 kurarinol 5,50 curcumin 6,51 maltol-3-O-(4′-O-cis-p-cumaroyl-6′-O-(3-hydroxy-3-methylglu-

Figure 7. Acid−base-nucleophile triad in the active site of the enzyme.

D




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Figure 8. Structures of compounds 1−10.

taroyl)-β-glucopyranoside 7,52 and (−)-rosmarinic acid 8,53 and compounds 954 and 1055 from libraries (Figure 8). Other strategies were based on molecular modeling, pharmacophore hypotheses, and 3D-QSAR models, and virtual screening. Literature survey revealed that several sortase inhibitors including diarylacrilonitriles, aryl(β-amino)ethyl ketones, rhodanines pyridazinones and pyrazolethiones, morpholinobenzoate derivatives, dihydro-β-carboline, benzo[d]isothiazol3(2H)-one-adamantane amine derivatives, and 3,6-disubstituted triazolothiazoles were obtained by these latter strategies. 4.1. Diarylacrylonitriles. High-throughput screening (HTS) is a widely used technique in the search of new SrtA inhibitors because it allows the identification of active molecules among tens of thousands of screened compounds.56 A biochemical assay was combined with fluorescence resonance energy transfer (FRET) to measure SrtA activity by monitoring the cleavage of a fluorescence resonance energy transfer substrate between the Thr and Gly residues.57,58 Oh et al.59 screened a library of 1000 small molecules using recombinant SrtA and identified methyl(2E)-2,3-bis(4methoxyphenyl)prop-2-enoate 11 (Figure 9) as a promising hit with an IC50 value of 231 μM. The authors synthesized a series of derivatives with the aim of understanding the structure−activity relationship (SAR) of this novel class of inhibitors. In particular, they investigated the influence of the methyl ester group by replacing it with a

carboxylic acid, a primary amide, or a nitrile group, and the importance of the double bond by examining the corresponding hydrogenated derivatives. The resulting compounds were inactive or poorly active with IC50 values against SrtA up to 1000 μM. Finally, they studied the influence of the double bond configuration discovering that (Z)-diarylacrylonitrile 12 was 7− 8-fold more potent against SrtA than derivative 11, with an IC50 value of 28 μM (Figure 9). In this class of compounds, the presence of a nitrile group and the Z double bond configuration represented structural features essential for SrtA inhibition. Among the diarylacrylonitriles synthesized and tested for SrtA inhibition, (Z)3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) 13 (Figure 9) was the most active compound with an IC50 value of 9.2 μM. Kinetic studies on derivative 13 demonstrated that it behaved as a competitive inhibitor with a Ki of 6.81 ± 0.41 μM and bound the enzyme in a reversible way. Molecular modeling studies59 predict that (Z)-2,3-diphenylacrylonitriles inhibit SrtA through strong lipophilic interactions between the phenyl rings of 3 and the alkyl side chains of Val193, Trp194, Ala92, Ala104, Leu169, Val168, and Ile182 forming the hydrophobic binding pocket in the active site (Figure 10). No interaction between the ligand and Cys184, which is crucial for transpeptidation activity, was identified even if the methoxy group of compound 13 is close to Cys184. However, the nitrile group of compound 13 forms a hydrogen bond with the catalytic residue Arg197 which plays an important role in the enzymatic mechanism.45 The authors have also extended the analysis of the pharmacological profile and mechanism of action of (Z)-2,3diphenylacrylonitriles in two additional studies.24,60 In particular, they explored the effects of three doses of compound 13 (4, 20, and 100 mg/kg) administered to Balb/c mice infected with 107 CFU of Newman wild-type S. aureus.60 Intravenous

Figure 9. Structures of compounds 11−13. E




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IC50 values (4.8 μM and 5.6 μM, respectively) for the inhibition of the SrtA homologue from B. anthracis. Kinetic analysis and mass spectrometry suggested a noncompetitive inhibition by an irreversible covalent modification of the thiol group of Cys184 via an elimination−addition mechanism. Aryl(β-amino)ethylketones 14 (Figure 12) undergo deprotonation by an active site base forming an enolate that is stabilized by the guanidinium of the conserved arginine. This enolate, after β-elimination, undergoes a nucleophilic attack on the vinyl group of the aryl vinyl ketone by the sulfur of the thiolic group of Cys184. This Michael-type addition inactivates the sortase (Figure 12). 4.3. Rhodanines, Pyridazinones, and Pyrazolethiones. Another class of potent SrtA inhibitors were obtained by Suree et al. using high-throughput screening.62 These authors screened about 30,000 compounds with a modified fluorescence resonance energy transfer assay to identify promising small molecules inhibitors that might be developed as antiinfective agents. The assay was performed using two criteria to calculate the percentage of inhibition of each compound in the library: the initial velocity of product formation and the endpoint analyses of product formation. Among >30,000 molecules, three compounds (17, 18, and 19; Figure 13), belonging to the families of rhodanines, pyridazinones, and pyrazolethiones were identified as leads. Compounds 17, 18, and 19 inhibited SrtA in a reversible fashion with IC50 values of 3.7 μM, 4.5 μM, and 5.2 μM, respectively. To improve the inhibitory activity of these leads, SAR analysis was performed using both purchased and synthesized analogues. In particular, among the rhodanine series, the 2,4dimethyl groups and the 2-OH group of compound 17 were shown to be fundamental for SrtA inhibition, their removal resulting in a 10-fold reduction of activity. None of the chemical substitutions improved the activity of lead 17, which is the most active of the rhodanines. In the pyridazinone series, replacement of −SH with −OH on the pyridazinone ring of compound 18 dramatically reduced the inhibitory activity, whereas the removal of 3-Cl on the phenyl ring significantly increased the potency. This generated derivative 20, which showed an IC50 value of 0.20 μM. The SAR of pyrazolethiones showed that the replacement of the thione group with a ketone was detrimental, whereas the replacement of the phenyl group with a more electron-withdrawing pyridyl group or with a cyclohexyl enhanced or reduced the potency, respectively. The most potent compound of this class (compound 21) showed an IC50 value of 0.30 μM. Although the mechanism of action of these molecules is not yet clear, spectroscopic studies on compound 17 have shown a reversible inhibition of SrtA through a thiol−disulfide exchange reaction with the Cys184 of the enzyme. The reversibility of enzyme inhibition was first determined by measuring the activity of the enzyme−inhibitor complex immediately after dilution. The results showed a rapid reversibility for compound 17 (84% of enzyme activity recovered after dilution) and a lower reversibility for compounds 18 and 19 (50% and 58% respectively). These data were confirmed by mass spectrometry. Mass spectra of the saturated SrtA-inhibitor complex were recorded at 1, 48, or 96 h after formation. These spectra were similar to those obtained with SrtA alone, suggesting that ligands do not cause permanent modifications of SrtA. Many of the molecules described by Suree et al. inhibited the SrtA homologue from both S. aureus and B. anthracis without detrimental effect on bacterial viability, highlighting their

Figure 10. Interaction of inhibitor 13 with the sortase A active site.59

administration of compound 13 reduced mortality and decreased infection of the kidneys and joints. The rate of survival was lower with the highest dose of compound 13, which might have caused off-target or toxic effects. The inhibitory activity of compound 13 on SrtA was demonstrated in a sortase inhibition assay (9.14 μM), as well as in a fibronectin-binding assay, in which the compound was able to inhibit S. aureus adhesion to fibronectin.24 4.2. Aryl(β-amino)ethyl Ketones. Through an HTS on a library of 136,625 small molecules, Maresso et al. identified aryl(β-amino)ethyl ketones (AAEK) as promising SrtA inhibitors.61 In particular, 6,154 molecules displayed a percent of inhibition greater than 20%, and after the elimination of compounds that were reactive, genotoxic, promiscuous, and lacking drug-like properties, only 407 compounds were attained. These compounds were clustered into five chemical classes: aryl(β-amino)ethylketones, N-arylmaleamides and fumaramides, N,4-diaryl-2-aminothiazoles, 3-heteroatom-substituted (N-alkyl/aryl)pyrrolidine-2,5-diones, and substituted maleimide-furan Diels−Alder products. Of these, the aryl(βamino)ethylketones were the most active inhibitors of sortases from straphylococci and bacilli. Aryl(β-amino)ethylketones 14 (Figure 11) are Mannich bases with a propiophenonic or related heteroaromatic core


bearing a β-arylamino or β-dialkylamino substituent. Two compounds of this class, 3-(dimethylamino)-1-(thiophen-2yl)propan-1-one 15 and 1-(3,4-dichlorophenyl)-3(dimethylamino)propan-1-one 16 (Figure 11) were selected for further studies because of their good inhibition of sortase and limited inhibition of papaine. Compounds 15 and 16 (Figure 11) showed IC50 values of 47 μM and 15 μM, respectively, for SrtA of S. aureus, and lower F




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Figure 12. Mecanism of action of Aryl(β-amino)ethylketones 4.61


of the phenyl ring, whereas the N-aryl substituent and rhodanine ring mapped the two hydrophobic regions. The pyridazinone analogue 20 showed the maximum overlap with all features of the pharmacophore (Figure 14c). In particular, the thiol group, the nitrogen present at position 1, and the carbonyl oxygen of pyridazinone were mapped with the three H-bind acceptors, whereas the two hydrophobic features were mapped with the aromatic ring substituent and within the center of pyridazinone ring. Finally, the pyrazolethione 21 (Figure 14d) mapped only with two H-bond acceptor features: the sulfur atom of pyrazolethione ring and the nitrogen present at position 2. The pyrazolethione and the center of the aromatic rings covered the two hydrophobic features. COMFA and CoMSIA analyses for compounds 19, 20, and 21, each from three different series, were also performed

potential as promising antivirulence drug candidates. Rhodanines, pyridazinones, and pyrazolethiones have been also the object of a combined pharmacophore and 3D-QSAR study focused on the identification of 3D pharmacophore model for S. aureus SrtA. A comprehensive collection of steric and electronic characteristics essential for ligand−enzyme interaction was described, which could lead to the discovery of new antibacterial drug candidates.63 Among these, the most active compounds (17, 20, and 21; Figure 13), each from three different series, were utilized in generating the pharmacophore hypothesis. The pharmacophoric features extracted from the most active compounds of the three series include three H-bond acceptor and two hydrophobic regions as reported in Figure 14a. The rhodanine analogue 17 (Figure 14b) overlaps only two of three H-bond acceptor regions with its carbonyl oxygen and hydroxyl group G




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Figure 14. Pharmacophoric features (a) and compounds 17 (b), 20 (c), and 21 (d) overlapped on the pharmacophoric features.63

suggesting, inter alia, the significant interaction of the hydrogen bond donor group with the identified pharmacophoric features. 4.4. Morpholinobenzoate Derivatives. Morpholinobenzoate derivatives are another class of SrtA inhibitors identified by in silico virtual screening of commercial compound libraries. Chenna et al.64,65 first identified new morpholinobenzoates using flexible docking (FLEX software package) on the high resolution structure of recombinant S. aureus SrtAΔ59,66 to fit approximately 150,000 small molecules with molecular weight between 150 and 500 and ClogP < 5 into the enzyme active site. The top scoring compounds (108 of 150,000) were screened against S. aureus SrtAΔ59, using a FRET enzymatic assay, with the intent to confirm the reliability of the chosen model. Among the leads identified from the screen, only the 7.4% (8 of 108 molecules) exhibited an in vitro inhibitory activity with IC50 values ranging between 75 and 400 μM. Compound 22 showed the greatest potency (Figure 15) with an IC50 value of 75 μM. The FlexX docking model revealed several critical interactions of compound 23 (Figure16) within the active site of the enzyme. In particular, its morpholine ring oxygen atom interacts with amide backbone NH of Trp194 with a

Figure 16. Interaction of inhibitor 23 with the sortase A active site.64 Reprinted with permission from ref 64. Copyright 2008 Elsevier.

hydrogen bond, the carboxylic acid of the middle phenyl ring forms a salt bridge interaction with Arg197, and the amide NH and the CO groups of the linker form two hydrogen bonds with Glu105 and Ser116, respectively (Figure 16). The replacement of the furan ring with a thiophene ring leads to the more active compounds 23 (Figure 15), which showed an IC50 of 58 μM. Similarly, methyl ester analogues showed improved inhibition as compared to the corresponding acids derivatives. In addition, the double bond appears to be necessary, as shown by the loss of activity after saturation of the double bond or its substitution with a triple bond. The authors also investigated the stereochemistry of the double bound that was critical for the inhibitory activity. Replacement of the E double bond with a Z double bond resulted in a reduction of activity in most of the cases. A recent study,67 based on a dataset of 34 novel S. aureus sortase A inhibitors 24−31 (Figure 17) with available IC50 data, allowed the authors to develop a ligand-based pharmacophore

Figure 15. Structures of compounds 22−23. H




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Figure 18. Pictorial representation of the pharmacophore model developed in the present study:67 (a) pharmacophore hypothesis and distances in Å between pharmacophoric sites; (b) compound 24a fitted into the pharmacophore model.

hypothesis and to derive a 3D-QSAR model for morpholinobenzoate derivatives.

Results led to the identification of a pharmacophore model, denoted as A2D3R7R8, which consists of one hydrogen bond I




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Figure 19. 3D-QSAR results: favorable substitutions on morpholinobenzoato derivatives.


increase in SrtA inhibitory activity. Positive effects are achieved with an electron withdrawing group and/or hydrogen bond donor groups in an area close to the H of −CONH (Figure 19, blue). Finally, for the morpholinobenzoic acid moiety (Figure 19, green), two substitution patterns should be considered. An increased activity might be obtained with hydrogen bond donor groups at position 2, hydrophobic groups at position 3, and electron donating groups around the N atom of the morpholine ring and/or with hydrogen bond acceptors or donors around the oxygen of the hydroxyl group, and hydrophobic groups around the carbonyl group of benzoic acid. 4.5. Dihydro-β-carboline. Chemical investigation of the tropical marine sponge Hyrtios sp led Lee et al.68 to the discovery, together with the known metabolites 32−37, of a

acceptor (A), one hydrogen bond donor (D), and two aromatic rings (R) (Figure 18a). Compound 24a, the most active of the whole set, well fits in this pharmacophore model, as shown in Figure 18b. In particular, 3D-QSAR data has helped to gain new insights into the structural requirements of S. aureus SrtA. Three welldefined areas of morpholinobenzoato derivatives were highlighted as crucial points for the introduction of substituents that can promote the SrtA inhibitory activity: (i) the thiophene ring, (ii) the amido group, and (iii) 2-morpholinobenzoic acid moiety (Figure 19). In the thiophene area (Figure 19, red), the presence of an electron withdrawing group, such as NO2, Cl, F, and/or hydrophobic groups at the 1, 2, and 4 positions will result in an J




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Figure 21. Structures and activities of compounds 42−44.

21) with an IC50 of 6.11 μM against SrtA in the biochemical assay. Furthermore, NMR experiments demonstrated its ability to bind the active center with high velocity in an irreversible fashion. These experiments also confirmed the reactivity toward the Cys184 side chain, which forms a covalent bond between the sulfur atom of the active site cysteine and the benzothiazol group of 42. The NMR structure of the enzyme complexed with inhibitor 42 showed how the benzo[d]isothiazol-3(2H)-one heterocycle, the adamantyl moiety, and the linker connecting these two groups are fundamental for binding and selectivity of the inhibitor. In particular, the benzo[d]isothiazol-3(2H)-one heterocycle is involved in the formation of a covalent bond with the enzyme; the adamantyl moiety fits into a hydrophobic pocket interacting with residues Ile182, Ile199, Val166, Val168, and Leu169; and the linker correctly positions the benzo[d]isothiazol-3(2H)-one heterocycle and the adamantane, and forms a hydrogen bond with Arg197 (Figure 22). A preliminary hit-to-lead optimization was performed starting from compound 42, by synthesizing a series of 15 compounds that exhibited combinations of substitutions in the adamantyl moiety, substitutions in the benzo[d]isothiazol3(2H)-one heterocycle, and variations of the linker. The resulting compounds exhibited high SrtA inhibition potency with IC50 values in the range 3.39−7.06 μM, but they showed cytotoxicity against mammalian fibroblast NIH 3T3, which might be due to off-target effects. On the basis of improved in vitro activity, good MIC values, cytotoxicity, and an advantageous selectivity index, derivatives 43 and 44 (Figure 21) can be considered as promising lead candidates for the development of novel antivirulence agents. 4.7. 3,6-Disubstituted Triazolothiazoles. The new class of 3,6-disubstituted triazolothiazole compounds as SrtA inhibitors was discovered by Zhang et al. in 201455 based on

new alkaloid, 6-hydroxydihydro-β-carboline 38 (Figure 20) endowed with inhibitory activity against isocitrate lyase (ICL) of Candida albicans. Further studies69 led the same authors to discover that 6hydroxydihydro-β-carboline 38 (Figure 20) inhibited SrtA activity with a potency in the high micromolar range (IC50 value = 290 μM). Starting from this lead, six new synthetic analogues of indole-type natural product analogues showing inhibitory activity against S. aureus SrtA greater than that of the reference compound, p-hydroxymercuricbenzoic acid (IC50 value =124 μM), were identified. Among the synthesized compounds, the best inhibitors were the indole analogues 39 and 40, and the dihydro-β-carboline 41 (Figure 20) with IC50 values of 61, 69, and 25 μM, respectively. As typically observed with SrtA inhibitors, compounds 39−41 had no effect on bacterial growth showing a very high MIC value against S. aureus.69 4.6. Benzo[d]isothiazol-3(2H)-one-adamantane amine derivatives. Zhulenkovs et al.70 identified a series of benzo[d]isothiazol-3(2H)-one-adamantane amine derivatives as a new class of SrtA inhibitors. In particular, a drug-like library of 50,240 compounds was selected by filtering the entire 1.6-million-compounds collection of Enamine (Enamine Ltd., Ukraine) to exclude compounds not eligible for drug discovery and compounds with reactive groups and toxicophores. The selected compounds were screened against the recombinant SrtAΔN59-6His in the FRET-based assay by validating the hits by NMR spectroscopy to exclude false positives and to study the binding of the inhibitor to the active site of SrtA. Among 41 compounds selected by the HTS assay, 11 compounds showed no binding to sortase during the HSCQ NMR experiments and were discarded. The best hit in this study was the N-(adamantan-1-yl)-2-(3oxo-2,3-dihydro-1,2-benzothiazol-2-yl)-acetamide 42 (Figure K



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inhibitory activity. Starting from the hit 46, 14 different compounds including 47, 48, and 49 (Figure 23) were generated. The best activity was displayed by compound 49 with an IC50 value of 9.3 μM, which represents a 4-fold improvement over hit 46. Compound 49 inhibited the enzyme in a concentration-dependent manner with 10.8−93.6% inhibition at 6.25 and 50 μM, respectively. The mechanism of sortase inhibition was also studied with surface plasmon resonance (SPR) and circular dichroism (CD), and data suggested that compound 49 acts as a reversible inhibitor with a KD of 8.8 μM and does not covalently modify the active site cysteine of sortase. Inhibition of sortase activity in staphylococci was also evaluated for compound 47, which blocked P2 precursor cleavage by SrtA, thereby preventing the incorporation of surface proteins (SpA) into the staphylococcal envelope. Other significant effects are the reduction of staphylococcal association with fibrinogen, important in the pathogenesis of bloodstream infections, and a slight reduction in the abundance of staphylococcal binder of Ig (Sbi) with a mechanism that has not be clarified as yet. The efficacy of compound 49 as anti-infective agent in preventing S. aureus bloodstream infections was highlighted by in vivo studies on BALB/c mice. In particular, mice (n = 15) were inoculated by i.p. injection with a dose of 40 mg/kg body weight of compound 49 at 12-h intervals for 5 days. Four hours after the first injection, animals were infected with i.v. injection with 1 × 107 CFU S. aureus Newman, and survival was recorded. More than half (8/15) of compound 49-treated mice survived staphylococcal bacteremia.


Figure 22. NMR structure of inhibitor 42 within the binding pocket.70 Reprinted with permission from ref 70. Copyright 2014 Elsevier.

in silico screening of a drug-like library of 300,000 compounds by combining scaffold hopping and molecular docking. Starting from the known inhibitor Topsentin A 4548 (Figure 23) taken as a model ligand, and using the SrtA−substrate complex to model the enzyme active site as a target for computational screening, they selected 105 compounds from the library for experimental validation on the purified recombinant sortase, SrtA ΔN24. The percent inhibition of sortase activity was first evaluated at 100 μM; then, IC50 values were calculated for the compounds showing a percent of inhibition greater than 50%. In order to determine whether the identified inhibitors also blocked sortase-catalyzed transpeptidation, the authors used a HPLC and MS assay to quantify SrtA cleavage of the LPATG substrate. Using this technique, compound 46 (Figure 23) was identified as hit compound with an IC50 value of 37.7 μM for S. aureus SrtA. A synthetic optimization of the 3,6-disubstituted triazolothiadiazole scaffold was also performed to improve its

5. CONCLUSIONS AND FUTURE DIRECTIONS The search for alternative strategies to conventional antibiotics in the struggle against pathogens and antibiotic resistance is a

Figure 23. Structures and activities of compounds 45−49. L




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conformational rearrangement, favored by substrate-induced activation, must occur in the side chains of His120 and Cys25 of SrtA. This difference could be advantageous in order to obtain new selective inhibitors of SrtA and prevent other proteolysis reactions. A major end point in the development of SrtA inhibitors is to generate drugs with high potency because most of the current compounds are active in the high micromolar range. Studies of SAR and 3D-QSAR, pharmacophore identification, and molecular docking are instrumental to reach this goal. Chemical modification of 6-hydroxydihydro-β-carboline 38 (IC50 value of 290 μM; see section 4.5) led to compound 41, with a 10-fold higher potency (IC50 value of 25 μM). Another example is provided by benzo[d]isothiazol-3(2H)-one-adamantane amine derivatives (section 4.6). Compound 42 was the best hit in the HTS assay with an IC50 of 6.11 μM. Structural modifications of compound 42, driven by molecular modeling, led to compounds 43 and 44, with IC50 values of 3.80 and 4.22 μM, respectively. Finally, 3,6-disubstituted triazolothiazoles (section 4.7) represent another class of SrtA inhibitors for which an improvement of activity was obtained. Compound 46 was identified as a hit compound with an IC50 value of 37.7 μM for S. aureus SrtA. Synthetic optimization of the 3,6disubstituted triazolothiadiazole led to compound 49 with an IC50 value of 9.3 μM, which represents a 4-fold improvement over hit 46. Most of the reviewed studies, however, are based on the identification of SrtA inhibitors by using enzymatic models, and only in few cases have the hit compounds been investigated in vivo or in vitro. The efficacy of the most promising SrtA inhibitors needs to be comprehensively evaluated in in vivo models of infection, including examination of survival rates, sepsis, abscess formation, septic arthritis, endocarditis, etc., prior to supporting an antivirulence role for each inhibitor that is worthy of further development into a novel therapeutic agent. In vitro evaluation of hit compounds on live bacterial cells, in terms of inhibition of biofilm formation and/or bacterial adhesion, should constitute a minimum requirement for the initial assessment of antivirulence activity of SrtA inhibitors following the demonstration of inhibitory activity in biochemical assays.20 The discovery of antivirulence drugs alternative to conventional antibiotics, despite its high importance, is at its infancy in terms of pharmaceutical development, and new in-depth studies are urgently needed. In addition, virulence attenuation as a strategy to combat bacterial infections requires a good host immune response for bacterial clearance because the virulenceattenuated pathogens will need to be successfully cleared by the host’s immune system. This is a point of weakness of antivirulence drugs because these drugs are not expected to be effective when given as monotherapy in immunecompromised patients. This challenge might be faced perhaps by exploring combination therapies with antivirulence and immune stimulating agents. The possibility of using novel agents that target virulence mechanisms and biofilm formation (antivirulence agents) in combination with current antibiotics represents a new avenue for the treatment of chronic bacterial infections and, again, could contribute to solving the problem of antibiotic resistance, which is recognized by the WHO as one of the most important global challenges of our time.

very considerable issue. Exploring alternative targets not associated with bacterial growth and cell death, but rather associated with virulence factors, could lead to the development of antivirulence drugs. Moreover, as opposed to conventional antibiotics, in response to which the evolution of resistance by the pathogens is advantageous and nearly unavoidable, in the case of antivirulence agents, the resistance is potentially costly and therefore less probable.71 Another interesting feature of antivirulence agents is that they could prevent infections without affecting the growth of beneficial microbiota, therefore being particularly helpful in the prophylaxis of MRSA infections without the classical adverse effects of antibiotics, for example, the rise of Clostridium dif f icile infections in a hospital setting.55 Several studies have confirmed that SrtA is a target not associated with bacterial growth but rather with virulence, and therefore, SrtA inhibitors do not kill bacteria but they reduce the virulence of pathogenic strains, thus restraining S. aureus infections. In this perspective, we have described the most promising small synthetic organic compounds that act as SrtA inhibitors and cater the potential to be developed into antivirulence drugs based on their activity at the low micromolar range. Two fundamental aspects must be highlighted with respect to the described SrtA inhibitors: (1) SrtA utilizes a catalytic cysteine residue common to several cysteine proteases. (2) The inhibition studies described thus far utilize the FRET-based cleavage of LPETG as a measure of sortase activity. Although well suited, this assay suffers several drawbacks. A high selectivity of SrtA inhibitors is fundamental to avoid off-target reactions which result in adverse effects, and special care must be taken to avoid false positive data in virtual screening. The search for novel antivirulence drugs acting as SrtA inhibitors is fueled by increasing knowledge of the structural and functional features of the enzymes. We predict that a new generation of SrtA inhibitors endowed with high potency and specificity will soon be available for clinical studies. This optimistic view is supported by the following considerations: (1) Some classes of described molecules (diarylacrylonitriles, rhodanines, pyridazinones, pyrazolethiones, and 3,6-disubstituted triazolothiadiazoles) are competitive noncovalent inhibitors, which, in some cases, do not interact with Cys184 and do not covalently modify the active site cysteine of sortase. These molecules are therefore good leads with therapeutic potential for further development. (2) Other classes of molecules (aryl(β-amino)ethyl ketones and benzo[d]isothiazol-3(2H)-one adamantanes) covalently modify the active cysteine residue of sortase. These molecules may alter the activity of other enzymes bearing an active cysteine residue, thereby causing cytotoxicity. However, even these irreversible inhibitors may show a favorable therapeutic window if their potency is increased by SAR. A good therapeutic efficacy might be reached with low doses of these drugs that do not cause toxic effects. Furthermore, the use of a filter for specificity measurements of papain activity in the high-throughput screening (see, for instance, aryl(β-amino)ethyl ketones) allowed one to select for moderate selective molecules that are good candidates as leads. However, the inability to react with the Cys-SH exerted by the sortase inhibitors having a Michael system does not eliminate the possibility that they might react with SH groups of other proteins producing off-target effects. As described above (see section 3), unlike the other cysteine protease, to enable sulfhydryl proton extraction and nucleophile attack, a M



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AUTHOR INFORMATION

biofilms. He concentrates on both synthetic organic compounds and natural products as antimicrobial peptides. He is an author of 62 publications in peer review journals. From 2001 to date, he has been teaching General Microbiology to the students of the School of Pharmacy of University of Palermo. He has acted as a reviewer for several journals in the applied microbiology field.

Corresponding Authors

*(S.C.) Phone: +39 091 23891920. E-mail: stellamaria. [email protected]. *(B.M.) Phone: +39 091 23891949. E-mail: benedetta. [email protected].

Giuseppe Daidone received a degree in Chemistry (cum laude) from University of Palermo in 1972. He conducted postgraduate research at the Institute of Pharmaceutical and Toxicological Chemistry of the University of Palermo where he was appointed associate Professor of Medicinal and Toxicological Chemistry. From 2001, he has been a Full Professor of Medicinal and Toxicological Chemistry at the Faculty of Pharmacy of the University of Palermo. His research focuses on the extension of the pyrazole chemistry and on the synthesis and biological evaluation of antiproliferative and antimicrobial agents.

Notes

The authors declare no competing financial interest.


Biographies Stella Cascioferro graduated in Pharmacy with honors in 1999, and she received her Ph.D. in Medicinal Chemistry in 2004 from the University of Palermo, under the supervision of Professor Demetrio Raffa, where she synthesized new 2-styrylquinazolin-4(3H)-ones with antileukemic activity. After obtaining her Ph.D., she joined the Physical and Theoretical Chemistry Laboratory, University of Oxford in the group of Professor Graham Richards. Her research interests include the design, synthesis, and biological evaluation of heterocyclic compounds as antitumoral, antinfective, and antiinflammatory agents. She is currently a Senior Scientist in the Department of STEBICEF, University of Palermo.

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ACKNOWLEDGMENTS We wish to express our sincere thanks to Profesoor Ferdinando Nicoletti for many helpful hints and a great deal of patience in reviewing this document.

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Demetrio Raffa received his Laurea Degree in Chemistry from the University of Palermo (Italy) in 1981. Afterward, he spent some months (November 1981−April 1982) in the Department of Organic Chemistry, University of Palermo. In 1983, he joined the Faculty of Pharmacy of Palermo as a Researcher. He worked from 07.09.1987 to 07.07.1988 as a Post Doctoral Fellow funded by CNR at the Institut Pasteur and Hopital St. Antoine, Paris. At present, he is Associate Professor of Medicinal Chemistry and carries on his research at the Department of Pharmaceutical Chemistry and Technology. He his an author and coauthor of 50 scientific papers mostly published in peerreviewed international journals. His research field is the design and synthesis of new heterocycles with antiinflammatory, analgesic antimicrobic, and antitumoral activity.

ABBREVIATIONS USED AAEK, aryl(β-amino)ethyl ketones; Balb/c, albino laboratorybred strain; CFU, colony forming units; Clf and Clf, clumping factors; COMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarity index analysis; Can, collagen-binding protein; 3D-QSAR, quantitative structure−activity relationship; DMMA, (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile; ESKAPE, Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterobacter; FLEX, flexible docking; Fnbp, fibronectin binding protein; FRET, fluorescence resonance energy transfer; HTS, high-throughput screening; ICL, isocitrate lyase; HSCQ NMR, heteronuclear single quantum coherence spectroscopy nuclear magnetic resonance; LPXTG, leucine, proline, any amino acid, threonine and glycine; MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MSCRAMMs, microbial surface components recognizing adhesive matrix molecules; SAR, structure−activity relationship; Sdr, serine-aspartate repeat proteins; Spa, protein A, SrtA, sortase A; WHO, World Health Organization

Benedetta Maggio obtained in 1983 her degree (cum laude) in Pharmacy from the University of Palermo. At the beginning of her career, she was a Technical Assistant in the Department of Medicinal Chemistry and Pharmaceutical Technology of the University of Palermo. From 1993, she was appointed University Researcher in Medicinal Chemistry at the Faculty of Pharmacy of University of Palermo. From the Academic year 2002/2003 to date, she is Professor of Drug Analysis at the School of Basic and Applied Sciences of the University of Palermo. Her scientific activity has been focused on the design synthesis and biological evaluation of compounds with antiproliferative, antimicrobial, and antiinflammatory activity.

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REFERENCES

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